Despite tremendous advances in cancer research over many years, cancer is still the second leading cause of death worldwide. For cancer treatment, extensive studies on the mechanisms thereof have revealed many specific molecular targets. Practically, cancer-targeted therapies have been very successful, particularly in the case of kinase inhibitors. Protein kinases are enzymes that catalyze the phosphorylation of hydroxy groups located in tyrosine, serine, and threonine residues of proteins, and play an important role in signal transduction of growth factors inducing cell growth, differentiation, and proliferation.
A signal transduction pathway should maintain a good balance between turning on and off so as to maintain homeostasis. However, mutations or overexpression of specific protein kinases disrupt the signal transduction pathway in normal cells (mainly, conditions wherein signal transduction in the body is continued), thus inducing various diseases such as cancer, inflammation, metabolic diseases, brain diseases, etc.
It is assumed that 518 kinds of human protein kinases exist, which correspond to about 1.7% of all human genes (Manning et al, Science, 2002, 298, 1912), and they are largely divided into tyrosine protein kinases (90 or more kinds) and serine/threonine protein kinases. Tyrosine protein kinases may be divided into 58 kinds of receptor tyrosine kinases that are classified into 20 subfamilies, and 32 kinds of cytoplasmic/non-receptor tyrosine kinases classified into 10 subfamilies. The receptor tyrosine kinases have a domain capable of accepting growth factors on the cell surface, and an active region capable of phosphorylating tyrosine residue in the cytoplasm. If a growth factor is bound to the growth factor receptor site on the cell surface of the receptor tyrosine kinase, the receptor tyrosine kinase forms a polymer and the tyrosine residue of cytoplasm is self-phosphorylated. Further, through sequential phosphorylation of the lower series of proteins, signal transduction is progressed into the nucleus, and ultimately, transcription factors inducing cancer are overexpressed.
Raf is serine/threonine (Ser/Thr) protein kinase, and plays a role for transducing signals sent by growth factor receptors activated in the cell membrane into the nucleus. A mitogen-activated protein kinase (MAPK) signal transduction pathway is essential for cell proliferation/differentiation/survival/apoptosis, etc., and is formed by the sequential phosphorylation of largely three kinases {MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK}. Raf is a MAPK kinase kinase (MAPKKK), MEK is a MAPK kinase (MAPKK), and extracellular signal-regulated kinase (ERK) is a MAPK. If a receptor is activated, a small GTP-binding protein Ras is activated, and through the sequential phosphorylation of Raf-MEK-ERK, MAPK signal transduction into the nucleus is achieved.
Meanwhile, a Ras oncogene that always maintains an activated state (particularly, k-Ras) is closely related to the induction of solid cancer such as pancreatic cancer (about 90%), rectal cancer (about 45%), liver cancer (about 30%), non-small cell lung cancer (about 35%), kidney cancer (about 10%), etc. If Raf-1 binds with activated Ras and the 338th serine of Raf-1 is phosphorylated (Avruch, J., Recent Progress in Hormone Research, 2001, 56, 127), Raf-1 is activated. To the contrary, if the 14-3-3 protein binds to Raf-1 of which 259th serine is phosphorylated, Raf-1 is inactivated.
The three subclasses of Raf protein (A-Raf, B-Raf, and C-Raf/Raf-1) have three conserved regions (CR1, CR2, and CR3) in the N-terminal control domain and C-terminal kinase domain (Tran et al., J Biol Chem, 2005, 280, 16244; Wellbrock, C., Nature Reviews Molecular Cell Biology, 2004, 5, 875).
The tissues where the three subclasses of Raf protein are expressed are different. C-Raf is expressed in substantially all tissues, A-Raf is mainly expressed in urogenital (kidney, uterine, and prostate) tissues, and B-Raf is mainly expressed in nervous, spleen, and hematopoietic tissues (Jaiswal, R. K. et al., J. Biol. Chem., 1966, 271, 23626). The overexpression of C-Raf is observed at about 50% in kidney cancer (renal cell carcinoma) and at about 100% in liver cancer (HCC), without expression of oncogenic mutant species.
Vascular endothelial growth factor receptors (VEGFR) are receptor tyrosine kinases (RTK), and are important modulators of angiogenesis. VEGFR is involved in angiogenesis, lymphangiogenesis, and homeostasis, and also has an important influence on neurons. VEGF is mainly produced in vascular endothelial cells, hematoblasts, and stromal cells by hypoxia and the stimulation of cell growth factors such as TGF, interleukin, and PDGF. VEGF binds to VEGF receptors (VEGFR)-1, (VEGFR)-2, and (VEGFR)-3, and each VEGF isoform binds to specific receptors to induce the formation of homozygotes or heterozygotes of the receptors, and then activates each signal transduction pathway. The signal specificity of VEGFR is controlled more minutely by coreceptors such as neurophilin, heparan sulfate, integrin, cadherin, etc.
The biological function of VEGF is mediated through type III RTK, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4). EGFR is closely related to Fms, Kit, and PDGFR, and each VEGF binds to specific receptors, wherein VEGF-A binds to VEGFR-1, VEGFR-2 and receptor heteromers, while VEGF-C binds to VEGF-2 and VEGFR-3. Further, PIGF and VEGF-B exclusively interact with VEGFR-1, and VEGF-E interacts only with VEGFR-2. VEGF-F variants interact with VEGFR-1 or VEGFR-2. VEGF-A, VEGF-B, and PIGF are preferentially required for angiogenesis, while VEGF-C and VEGF-D are essential for lymphangiogenesis. New blood vessels feed nutrients and oxygen to tumors and provides a passage of cancer metastasis, and thus are essential for proliferation and metastasis. Angiogenesis normally maintains a balance in the body by the mutual regulation of angiogenic stimulators and angiogenic suppressors, but if such a balance is broken as in cancer cells, the receptor VEGFR is activated by vascular endothelial growth factor (VEGF) that has the greatest influence on vascular endothelial cells.
Among various action mechanisms, inhibitors suppressing VEGF receptor tyrosine kinase using low molecular synthetic material are being variously studied and developed, and most of them have a possibility of being commonly used for solid tumors and suppress angiogenesis activated only in cancer cells, and thus have advantages in that medicinal effects can be expected with relatively few side effects.